Melatonin Improves the Resistance of Oxidative Stress-Induced Cellular Senescence in Osteoporotic Bone Marrow Mesenchymal Stem Cells

Figure 1

Evaluation of the resistance of BMMSCs to oxidative stress-induced premature senescence. BMMSCs from sham-operated (sham) and ovariectomized (OVX) rats were exposed to hydrogen peroxide (H₂O₂) ranging from 50 μM to 200 μM for 2 h and cultured for an additional 72 h. (a, b) Senescence-associated β-galactosidase (SA-β-gal) staining was performed to label senescent cells.  μm. (c) The mRNA expression levels of P16, P21, P53, and Sirt1 were quantified using qRT-PCR. (e) The protein levels of P16, P21, P53, and SIRT1 were determined using Western blot assays. (f) After exposure to 100 μM of H₂O₂, BMMSCs were induced toward the osteogenic differentiation for 14 days. Representative images of mineralized extracellular matrix stained by Alizarin Red S (ARS).  μm. (g) Quantification of the stained mineral layers in H₂O₂-treated and untreated BMMSCs. The values shown were normalized to those of the untreated sham BMMSCs. (h) The mRNA levels of osteoblast-specific marker genes, including Runx2, Sp7, and Bglap, were quantified with qRT-PCR using Gapdh as the reference gene. Data are shown as the of six independent experiments () in SA-β-gal staining, four independent experiments () in ARS assays, four independent experiments () in qRT-PCR experiments, and three independent experiments () in Western blot assays. Statistically significant differences are indicated by or between the indicated groups; ^Φ or ^ΦΦ versus untreated sham BMMSCs; ^Ψ or ^ΨΨ versus untreated OVX BMMSCs.

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